The enzyme alpha-galactosidase (E.C. 3.2.1.22; alpha-D-galactoside galactohydrolase) catalyzes the hydrolysis of the terminal linked alpha-galactose moiety from galactose-containing oligosaccharides. These include, for example, the naturally occurring disaccharide melibiose (6-O-alpha-D-galactopyranosyl-D-glucose), the trisaccharide raffinose (O-alpha-D-galactopyranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-2)-beta-D-fructofuranoside) and the tetrasaccharide stachyose (O-alpha-D-galactopyranosyl-(1-6)-O-alpha-D-galactopyranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-2)-beta-D-fructofuranoside). Alpha-galactosidases have potential use in various applications, and some examples are described by Margolles-Clark et al. (“Three alpha-galactosidase genes of Trichoderma reesi cloned by expression in yeast”, Eur. J. Biochemistry, 240:104-111, 1996). They may hydrolyze alpha-galactose residues from polymeric galactomannans, such as in guar gum; modification of guar gum galactomannan with alpha-galactosidase has been used to improve the gelling properties of the polysaccharide (Bulpin, P. V., et al., “Development of a biotechnological process for the modification of galactomannan polymers with plant alpha-galactosidase”, Carbohydrate Polymers 12:155-168, 1990). Alpha-galactosidase can hydrolyze raffinose from beet sugar syrup, which can be used to facilitate the sugar crystallization from molasses, since the raffinose presents an obstacle to the normal crystallization of beet sugar (Suzuki et al., “Studies on the decomposition of raffinose by alpha-galactosidase of mold” Agr. Biol. Chem., 33:501-513, 1969). Additionally, alpha-galactosidase can be used to hydrolyze stachyose and raffinose in soybean milk, thereby reducing or eliminating the undesirable digestive side effects which are associated with soybean milk (Thananunkal et al., “Degradation of raffinose and stachyose in soybean milk by alpha-galactosidase from Mortierella vinacea” Jour. Food Science, 41:173-175, 1976). The enzyme can also remove the terminal alpha-galactose residue from other glycans, such as the erythrocyte surface antigen conferring blood group B specificity. This has potential medical use in transfusion therapy by converting blood group type B to universal donor type O (Harpaz et al. “Studies on B-antigenic sites of human erythrocytes by use of coffee bean alpha-galactosidase”, Archives of Biochemistry and Biophysics, 170:676-683, 1975; and by Zhu et al. “Characterization of recombinant alpha-galactosidase for use in seroconversion from blood group B to O of human erythrocytes”, Archives of Biochemistry and Biophysics, 327:324-329, 1996).
Plant Alpha-Galactosidases
Plant alpha-galactosidases from numerous sources have been studied and multiple forms of the enzyme have been described, such as in Keller F. and Pharr D. M., “Metabolism of Carbohydrates in Sinks and Sources: Galactosyl-Sucrose Oligosaccharides”, In: Zamski, E. and Schaffer, A. A. (eds.) Photoassimilate Partitioning in Plants and Crops: Source-Sink Relationships, ch. 7, pp. 168-171, 1996, Marcel Dekker Publ., New York. These can be classified into two broad groups, acid or alkaline, according to the pH at which they show optimal activity. Practically all studies of alpha-galactosidases have dealt with the acidic forms of the enzyme and these play important roles in seed development and germination. Alpha-galactosidases with optimal activity at alkaline pH are uncommon in eucaryotic organisms.
Alpha-galactosidases which show preferred activity to the disaccharide melibiose are often referred to as melibiases. These may have optimal activity at alkaline pH but are relatively specific to melibiose, with only little activity and low affinity to the trisaccharide raffinose. In addition, they characteristically function as a multimeric protein. For example, the bacterial alpha-galactosidase that has been described from Bacillus stearothermophilus (Talbot, G. and Sygusch, J., “Purification and characterization of thermostable beta-mannanase and alpha-galactosidase from Bacillus stearothermophilus”, Applied and Environmental Microbiology, 56:3503-3510, 1990) has over a 15-fold higher activity with melibiose, as compared to raffinose and functions as a trimer. The alpha-galactosidase described from Escherichia coli K12 similarly has only about 4% of the activity with raffinose as compared to melibiose, with Km values of 60 mM and 3.2 mM, respectively, in addition to functioning as a tetrameric protein (Schmid and Schmitt, “Raffinose metabolism in Escherichia coli K12: purification and properties of a new alpha-galactosidase specified by a transmissible plasmid”, Eur. J. Biochemistry, 67:95-104, 1976). Similarly, the enzyme from Pseudomonas fluorescens H-601 (Hashimoto, H. et al., “Purification and some properties of alpha-galactosidase from Pseudomonas fluorescens H-601”, Agric. Biol. Chem., 55:2831-2838, 1991) has relative Km values for raffinose and melibiose of 17 and 3.2 mM, respectively, and functions as a tetramer.
There are obvious advantages to the use of a monomer protein with the desired enzyme activity, as compared to multimeric proteins. This has clearly been shown, for example, with the alpha-galactosidases from mung bean seeds (del Campillo, E., et al., “Molecular properties of the enzymic phytohemagglutinin of mung bean”, J. Biol. Chem. 256:7177-7180, 1981) in which the tetrameric form of the enzyme disassociated into the monomeric form during storage, and this was accompanied by loss of activity.
The galactosyl-sucrose sugars, stachyose and raffinose, together with sucrose, are the primary translocated sugars in the phloem of cucurbits, which includes muskmelons, pumpkins and cucumber. The very low concentrations of raffinose and stachyose in fruit tissues of muskmelon suggest that galactosyl-sucrose unloaded from phloem is rapidly metabolized, with the initial hydrolysis by alpha-galactosidase, as described in “Cucurbits”, Schaffer, A. A., Madore, M. and Pharr, D. M., In: Zamski, E. and Schaffer, A. A. (eds.) Photoassimilate Partitioning in Plants and Crops: Source-Sink Relationships, ch. 31, pp. 729-758, 1996, Marcel Dekker Publ., New York.
P.-R. Gaudreault and J. A. Webb have described in several publications, (such as “Alkaline alpha-galactosidase in leaves of Cucurbita pepo”, Plant Sci. Lett. 24, 281-288, 1982, “Partial purification and properties of an alkaline alpha-galactosidase from mature leaves of Cucurbita pepo”, Plant Physiol., 71, 662-668, 1983, and ‘Alkaline alpha-galactosidase activity and galactose metabolism in the family Cucurbitaceae”, Plant Science, 45, 71-75, 1986), a novel alpha-galactosidase purified from young leaves of Cucurbita pepo, that has an optimal activity at alkaline conditions (pH 7.5). In addition to the alkaline alpha-galactosidase, they also reported three acid forms of the enzyme, and distinct substrate preferences were found for the acid and alkaline forms. Raffinose was found to be the preferred substrate of the acidic forms. The alkaline form had high affinity (Km=4.5 mM) and high activity (1.58 μmol galactose formed per min per mg protein) only with stachyose. It had low affinity for (Km=36.4 mM) and low activity (0.14 μmol galactose formed per min. per mg protein) toward the trisaccharide raffinose but hydrolyzed melibiose so slowly that affinity and activity with melibiose was not calculated. Thus, the alkaline alpha-galactosidase reported by Gaudreault and Webb can be described as having activity at alkaline pH but only within a narrow spectrum of substrates. This is clearly a disadvantage, a wider substrate specificity affording more numerous practical applications.
A further characteristic of the alkaline alpha-galactosidase from young leaves of Cucurbita pepo is that alpha-D-galactose, the product of the enzymatic reaction, is a strong inhibitor of the enzyme's activity (Gaudreault and Webb, Plant Physiol., 1983, 71:662-668,), similar to many of the acid alpha-galactosidases. Geaudreault and Webb calculated that 6.4 mM galactose reduced the reaction velocity of alkaline alpha-galactosidase by 50%, in a reaction mixture containing 7.5 mM pNPG at pH 7.5. Such an inhibition by the product of the reaction (termed “product inhibition”), generally has important physiological significance in metabolism.
Gaudreault and Webb (among others) have suggested that the alkaline alpha-galactosidase, as the initial enzyme in the metabolic pathway of stachyose and raffinose catabolism, was important in phloem unloading and catabolism of transported stachyose in the young cucurbit leaf tissue. It is likely that alpha-galactosidase similarly plays an important role in the carbohydrate partitioning in melon plants, and may have possible functions for phloem unloading in fruits of muskmelon. Recently, alpha-galactosidase activity at alkaline pH has been observed in other cucurbit tissue, such as cucumber fruit pedicels, young squash fruit and young melon fruit. Results obtained by Pharr and Hubbard (“Melons: Biochemical and Physiological Control of Sugar Accumulation, In: Encyclopedia of Agricultural Science, vol. 3, pp. 25-37, Arntzen, C. J., et al., eds. Academic Press, New York, 1994) led them to suggest that stachyose degradation by alpha-galactosidase took place within pedicels of fruit of Cucumis sativus, especially in the regions where the pedicel joins the fruit. Recently, Irving et al. (“Changes in carbohydrates and carbohydrate metabolizing enzymes during the development, maturation and ripening of buttercup squash, Cucurbita maxima D. ‘Delica’”, J. Amer. Soc. Hort. Sci., 122: 310-314, 1997) reported the developmental changes in alpha-galactosidase activities, measured at acid and alkaline pH, in buttercup squash (Cucurbita maxima) fruit. They found that at anthesis, alkaline activity was higher than activity at acid pH and that both activities declined during fruit development. Chrost and Schmitz (“Changes in soluble sugar and activity of alpha-galactosidase and acid invertase during muskmelon (Cucumis melo L.) fruit development”. J. of Plant Physiology, 151:41-50, 1977) reported approximately similar activities of alpha-galactosidase at acid and alkaline pH in Cucumis melo fruit at the anthesis stage.
However, all of these studies were carried out using the non-specific artificial substrate, p-nitrophenyl alpha-galactopyranoside (pNPG), which indicates alpha-galactosidase activity but does not reflect either the physiological role of the particular enzyme forms, or, more importantly, the substrate specificity of the particular enzyme.
Indeed, it is well established that the artificial substrate pNPG often indicates a higher pH optimum for alpha-galactosidase activity than that which is observed with the natural substrates. For example, Courtois and Petek (“alpha-galactosidase from coffee bean”, Methods in Enzymology, vol. 8:565-571, 1966) state that “With alpha-phenylgalactoside (pNPG) one observes a pH optimum at pH 3.6, and a second more pronounced peak at pH 6.1. Toward other substrates (melibiose, raffinose, planteose and stachyose) the pH curve is flatter, with a maximum between 3.6 and 4.0”. Similar results were observed for the alpha-galactosidase of Vicia faba seeds (Dey, P. M. and Pridham, J. B., “Purification and properties of alpha-galactosidase from Vicia faba seeds”, Bioch. J., 113:49-54, 1969). Thus, the abovementioned observations of alkaline alpha-galactosidase enzyme activity in the fruit pedicel or fruit tissue, assayed with pNPG, give no indication as to the actual character of the catalytic activity measured, and cannot clearly distinguish such activity from previously described alpha-galactosidase.
While it had been thought that alkaline alpha-galactosidase may be confined to the cucurbit family, which includes the above mentioned squash, cucumber and melon plants, it has recently been shown by Bachmann et al. (“Metabolism of the raffinose family oligosaccharides in leaves of Ajuga reptens L.”, Plant Physiology 105:1335-1345, 1994) that Ajuga reptens plants (common bugle), a stachyose translocator from the unrelated Lamiaceae family also contains an alkaline alpha-galactosidase. This enzyme was partially characterized and found to have high affinity to stachyose. Also, leaves of the Peperomia camptotricha L. plant, from the family Piperaceae, show alpha-galactosidase activity at alkaline pH, suggesting that they also contain an alkaline alpha-galactosidase enzyme (Madore, M., “Catabolism of raffinose family oligosaccharides by vegetative sink tissues”, In: Carbon Partitioning and Source-Sink Interactions in Plants, Madore, M. and Lucas, W. J. (eds.) pp. 204-214, 1995, American Society of Plant Physiologists, Maryland). This indicates the possibility that alkaline alpha-galactosidases, including novel enzymes not previously described, may function in other plants that metabolize galactosyl-saccharides, in addition to the cucurbits. Similarly, Gao and Schaffer (Plant Physiol. 1999; 119:979-88, which is incorporated fully herein by reference) have reported an alpha galactosidase activity with alkaline pH optimum in crude extracts of tissues from a variety of species including members of the Cucurbit and Coleus (Lamiaceae) families.
The use of an acidic form of alpha-galactosidase in biotechnological and industrial applications presents problems. For example, the use of an acidic form of alpha-galactosidase to remove the galactose-containing oligosaccharides, which include raffinose and stachyose, from soybean milk presents a dilemma, as described by Thanaunkul et al., (“Degradation of raffinose and stachyose in soybean milk by alpha-galactosidase from Mortierella vinacea” Jour. Food Science, 41:173-175, 1976). The pH of soybean milk, which is 6.2-6.4, is well above the optimum pH range of the Mortariella vinacea enzyme, which is 4.0-4.5, as shown using the natural substrate melibiose. Lowering the pH of the soybean milk solution to conform to the acidic enzyme's pH optimum caused the soybean proteins to precipitate and left a sour taste to the milk.
The use of alpha-galactosidase with an acidic pH optimum for the removal of raffinose from beet sugar faces a similar problem. In Suzuki et, 1969, (“Studies on the decomposition of raffinose by alpha-galactosidase of mold” Agr. Biol. Chem., 33:501-513, 1969) the pH of the beet molasses had to be lowered to 5.2 with sulfuric acid in order for the Mortariella vinacea enzyme to function.
Similarly, seroconversion of blood type B to blood type O would benefit from an alpha-galactosidase that is active at neutral to alkaline pH. since the described procedure (Goldstein et al., “Group B erythrocytes enzymatically converted to group O survive normally in A, B, and O individuals” Science, 215:168-170, 1982) requires the transfer of centrifuged erythrocytes to an acidic buffer in order for the enzyme to function. Lowering the pH to the optimum for the coffee bean alpha-galactosidase caused the cells to be less stable and lysis to occur. Thus, the seroconversion is carried out at pH 5.6, “reflecting a compromise between red cell viability and optimal alpha-galactosidase activity”, as reported in Zhu et al. (“Characterization of recombinant alpha-galactosidase for use in seroconversion from blood group B to O of human erythrocytes”, Archives of Biochemistry and Biophysics, 327:324-329, 1996). Since the natural pH of blood is in the neutral to alkaline range (pH 7.3) an alpha-galactosidase with activity in this pH range would have obvious advantages.
An additional limitation on the industrial utility of the currently available alpha-galactosidases is that their activity is frequently inhibited by the product of the reaction, galactose. As an example, the reported alkaline alpha-galactosidase from Cucurbita pepo leaves (Geaudreault, P. R. and Webb, J. A. “Partial purification and properties of an alkaline alpha-galactosidase from mature leaves of Cucurbita pepo”, Plant Physiol., 71, 662-668, 1983) is strongly inhibited by alpha-galactose and it was calculated that 6.4 mM galactose reduced the reaction velocity by 50%.
Thus, there is a well-established need for an alpha-galactosidase with high activity at neutral to alkaline pH and with activity towards a broad spectrum of natural galactose-containing saccharides, particularly, but not exclusively raffinose.